Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: Implications for wild-type, V30M, and L55P amyloid fibril formation

Analytical ultracentrifugation methods were utilized to further characteriz e the acid denaturation pathways of wild-type. V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid poly neuropathy. Equilibrium and velocity methods were employed herein to charac terize the TTR quaternary structural requirements for amyloid fibril format ion. From neutral to slightly acidic conditions (pH 7.5-5.1), wildtype tran sthyretin (0.2-0.3 mg/mL, 100 mM KCI, 37 degrees C) exists as a tetramer an d is incapable of fibril formation. Under more acidic conditions (pH 5 to 3 .9), tetrameric wild-type TTR slowly dissociates to a monomer having an alt ernatively folded tertiary structure(s) that self-assembles at physiologica l concentration (0.2 mg/mL) into a ladder of quaternary structural intermed iates of increasing molecular weight. These intermediates appear to be on t he pathway of amyloid fibril formation, since they ultimately disappear whe n amyloid fibrils are observed. The V30M and L55P TTR variants exhibit simi lar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermedi ates to be readily observed by velocity methods. Partial denaturation and a ssembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amy loid protofilaments at pH 7.5 (37 degrees C) after several weeks of incubat ion, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP vari ants relative to wild-type TTR, which does not form amyloid or amyloid prot ofilaments under these conditions. This study establishes the key role of t he monomeric amyloidogenic intermediate and its self-assembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30 M, and L55P transthyretin amyloid fibrils.